Emerging Intestinal Microsporidia Infection in General Population in Jiroft District, Southeastern Iran: A Cross-sectional Study in 2013-2014.

Background
Microsporidia have been reported as the cause of opportunistic infections in immunocompromised patients in Iran and other countries. There is no data on prevalence of intestinal microsporidia in healthy population of Iran. This study aimed to provide preliminary data on the present status of microsporidia infection in the local healthy population in Jiroft, Kerman Province from southeastern Iran in 2013-2014.


Methods
Fresh stool samples were randomly collected from 418 residents in rural 209 (50%) and urban 209 (50%) areas of Jiroft. All of the collected samples were concentrated with conventional formalin-ether, stained with Ryan blue. Microscopic examination was performed with high magnification on each sample separately for the demonstration of microsporidia spores.


Results
Microsporidial spores were identified in 41 out of 418 (9.8%) samples including 16.41(39%) from rural areas and 25.41(61%) from urban areas. In general, there was no significant difference between sex, age, job, education, and contact with soil and livestock, water supply, gastrointestinal disorders and microsporidia infection among general population in Jiroft.


Conclusion
Intestinal microsporidia infection without clinical manifestations is prevalent in general population resident in southeastern Iran. Appropriate molecular methods are needed for microsporidia species identification.


Introduction
Microsporidia become known as one of responsive agents of opportunistic infections in patients with AIDS and other immunocompromised individuals (1). Microsporidian infections of human sometimes could cause a disease called microsporidiosis. Up to now, 8 genera and 14 species of microsporidia have known as causative agent of human infection (2). Microsporidia have the smallest known eukaryotic genomes that characterize all known species (3). Microsporidial genera such as Enterocytozoon, Encephalitozoon species including Encephalitozoon cuniculi, E. hellem, and E. intestinalis are the most common microsporidia associated with human disease (4). Although microsporidiosis has been identified in a broader range of human populations including travelers, children, the elderly, organ transplant recipients, patients with malignant disease and diabetes (5), Immunocompromised patient, especially HIV-positive population is considered more susceptible to parasite infections than other populations (6). The most common clinical symptoms among immunodeficient patients are chronic diarrhea, weight loss, systemic diseases and in some cases mortality, While immunocompetent persons often have mild or self-limiting disease (1,7). Microsporidian diarrhea has been reported in travelers because of an outbreak related with contaminated cucumbers (5). The microsporidian infections have been occurred using contaminated water supplies by wild and domestic animal or food that produced from farm animals, shows importance of this food and water zoonotic disease (7). The use of antiretroviral therapy for preventing opportunistic infection in HIV positive patient has decreased the rate of microsporidian infection, however, is more reported as self-limited diarrhea and other intestinal complications in non-HIV immunodeficient individuals and in healthy people (8). Asymptomatic microsporidiosis have been reported among immunocompetent people and seroprevalence data showed that human in exposes with infection (1,(9)(10)(11). Diagnosis of microsporidia is according to identification of spores by parasitological methods like chromotrope 2R, Calcofluor white (12) and molecular method (11,13) described before. Enterocytozoon bieneusi has been reported as an important cause of chronic diarrhea in persons infected with human immunodeficiency virus (HIV) in Iran and other countries (1,(13)(14)(15). Enterocytozoon intestinalis is also another cause of chronic diarrhea in HIV+ population and immunocompromised people (6,16). Encephalitozoon cuniculi is a microsporidia species most commonly recognized as a cause of renal, respiratory, and central nervous system infections in immunosuppressed patients (4). Wildlife species like foxes could be significant reservoirs of E. cuniculi infection for both domestic animals and humans (17), and findings of the E. cuniculi from human and rabbits from different regions of the China revealed zoonotic sources of infection involving rabbits to be important route of transmission in humans (18).
Recently researchers have been focused on emerging opportunistic infection in Iran. Some studies have been described the frequency of intestinal microsporidia and coccidian parasites in HIV positive patient [13,19,20] and liver transplant population from Iran (21). However, there is no data about prevalence of intestinal microsporidia in general population in Iran. Regarding increasing trend of immunocompromised patients like HIV+ patients and transplant recipients and cancer patients, identification of the microsporidia in clinical samples is important to treat and reduce dissemination risks among patients in Iran. This study aimed to provide preliminary information on the prevalence of microsporidia in the local general population in Jiroft, Kerman Province in Iran in 2013-2014.

Study area
This study was performed in Jiroft, south Kerman Province, and southern Iran (Fig. 1).

Fig. 1: Situation of Jiroft in Kerman Province in Iran
It is comprised of four districts, fourteen rural districts, and four towns. Jiroft has a population of 277748, among whom 121988 (36%) lived in urban areas while the rest resided in villages. The town of Jiroft is located approximately 248 km 2 southeast of Kerman. (https://en.wikipedia.org/wiki/Jiroft_County).

Samples Collection
In this cross-sectional study, 418 individuals including residents of rural and urban areas were enrolled to evaluate intestinal microsporidia infections during 2013-2014. Sample collection was done according to cluster random sampling. Information about the infection was collected by filling up questionnaire by people in the community. we designed some simple questions about the basic amenities available, the use of sanitary toilets, water supply including pipe, spring or well-drinking water, gender, age, education, job, gastrointestinal disorder and contact with soil and animals that may be source of infection. Fresh stool samples were collected from all individuals and examined by formalin-ether concentration. All of samples were examined for intestinal microsporidia infections in the department of Medical Parasitology, School of Public Health, Tehran University of Medical Sciences. Informed consent was taken from the participants and the study was approved by Ethics Committee of Tehran University of Medical Sciences, Iran.

Ryan blue staining method
Conventional formalin-ether was done for all samples, for detection of intestinal microsporidia parasites, thin slides were prepared from stool samples and after methanol fixation allow the slides to dry for 10 min and all of slides were stained according to Ryan blue staining method (22). Finally, slides were examined using light microscope with 1000-x magnification. The analysis was performed using SPSS ver. 18 (Chicago, IL, USA) and Chi-square.

Results
Overall 418 individuals were enrolled in this study; 229 (54.8%) were female and 189 (45.2%) were male and 209(50%) lived in rural and 209(50%) lived in urban areas of Jiroft. Transparent ovoid microsporidian spores were observed measuring 1-1.5 μm size with a background of blue staining fecal bacteria in positive stool samples with 1000 magnification (Fig. 2).  Although infection rate of microsporidial infection was more in people who had abdominal pain in this study than people without gastrointestinal discomfort, there was no statistical significance between gastrointestinal discomfort and microsporidia infection. In general, there was no significant difference between microsporidia infection and sex, age, job, education, contact with soil and livestock, water supply and gastrointestinal discomfort in this study.

Discussion
The prevalence of microsporidia infection was between 0%-50% in different areas using parasitological and molecular diagnostic method (23).
Prevalence of microsporidia has also been reported between 7 %-50% in patients with AIDS (24). There are few reports on microsporidian infection in HIV+ population and transplant recipient from Iran. In a study, 356 HIV+ patients were evaluated in Shiraz, Iran, eight with persistent chronic diarrhea were found to be positive for E. bieneusi (genotypes D and K) using microscopic examination and the nested PCR technique (13) and in another study E. bieneusi (genotype D) were detected in 6.81% of liver transplant children (21). In a study, 25 out of 81(30.86%) stool samples from HIV+/AIDS patients from Tehran were positive for intestinal E. bieneusi infection, by nested PCR and staining method. Although chronic watery or moderate diarrhea was existed in 13 (52%) of positive cases no statistically significant difference was found (19). However, in a few cases, infections in immunocompetent people also have been observed (25)(26)(27). The actual frequency of intestinal microsporidial infections in healthy people is unknown. Microsporidia still are, often overlooked and misdiagnosed because they are not specifically identified in most diagnostic labs, they are rather small, needed different staining methods and expert technologist (1,5).
Regarding improved diagnostic methods, microsporidia have become more described even in healthy people without any clinical manifestation (27)(28)(29). Present study is the first report on microsporidian infection in healthy people in Jiroft, south of Iran. In our study, prevalence of microsporidial infection was 9.8% in rural and urban areas by parasitological method. There was no significant relationship between infection and gastrointestinal disorders. The most infected intestinal microsporidia 12.41(29.3%) belonged to children aged 0-9 yr old but there was no significant difference (P-value: 0.7) between age and microsporidian infection in this study. In Spain, 17% of 60 HIVnegative patients were infected with microsporidia (26). The frequency of microsporidiosis in healthy people in Cameroon was noticeably high. In a study, 67.5% of the 126 healthy people were positive for microsporidiosis by calcofluor white and randomly selected samples were confirmed E. bieneusi (27). The frequency of E. bieneusi was 35.7%, in patient with HIV positive and tuberculosis coinfection, but this rate was only 24.0% among only tuberculosis patients. The infection rate among healthy people was significantly higher from those of both groups of tuberculosis positive patients. The most infection rate was among juvenile group, and the most infection degree (2.5) was in children (27).
Another report of such a high prevalence of microsporidian infections is related to Ugandan children with persistent diarrhea, of which 32.9% were shedding of E. bieneusi (28).
Prevalence of E. bieneusi infections among children aged between 3-36 months with diarrhea was17.4%, however, this rate was 16.8% in control group of children in Uganda and the highest infection was found in rainy seasons. There was no significant difference between E. bieneusi infection and low growth or low weight infants and acute diarrhea (29). In our study, the highest infection rate was found among children aged between 0-9 yr old. A high prevalence of microsporidia was found in immunocompetent people in a study.

Conclusion
Microsporidial infections in general population of southeast of Iran is prevalent. No signs and symptoms are observed in general population resident in the infected individuals. Appropriate molecular methods are needed for identification of microsporidia species.

Ethical considerations
The study was performed in compliance with current national laws and regulations. Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.